|摘要: ||高分子材料於藥物傳遞領域的發展，扮演著不可或缺的角色。本研究以含脂質高分子 poly(acrylic acid-co-distearin acrylate) (poly(AAc-co-DSA)) 為材料，發展出各式多功能智慧型高分子組裝作為治療 (therapeutic) 及造影試劑 (diagnostic agents) 的傳遞系統，用以改善藥物對腫瘤的傳遞及治療效率。欲發展智慧型藥物傳遞系統，必須能精確掌握高分子組裝的分子排列應答機制，才能設計出符合各式需求的藥物傳遞系統。|
本研究利用含脂質高分子 poly(AAc-co-DSA) 於水相中會自組裝的特性，製備出具酸鹼應答穿膜通道 (pH-responsive transmembrane channel) 的高分子液胞。藉由簡單調整液胞所處環境 pH或高分子組成，可有效調節液胞穿膜通道尺寸大小，對通過通道的物質大小進行篩選。我們發現高分子液胞的通道尺寸大小，會隨著液胞所處環境 pH 增加而擴大。另一方面，若製備液胞所用含脂質高分子的 DSA 含量愈高，則開啟液胞膜內穿膜通道所需的 pH 亦隨之增加，僅能允許分子量較小的水溶性分子通過液胞膜。加入少量的鈣離子與液胞膜上解離的 AAc 單元螯合，藉此穩定液胞結構及固定液胞通道的大小，進一步引發凝縮作用 (stacking effect)，減少液胞膜內由 polyAAc 所構成的立體障礙，使水溶性分子更易通過穿膜通道，降低通透液胞膜所需 pH。水溶性分子能通透高分子液胞主要是因為，液胞膜內緻密的分子排列會隨著液胞溶液 pH 增加而被破壞，進而擴大穿膜通道的尺寸大小。
隨著研究的進行，高分子液胞內分子組裝對 pH 的應答特性逐步被釐清，為進一步拓展高分子液胞於藥物傳遞及藥物控制釋放的應用性，粒徑為140 ~ 160 nm的奈米載藥高分子液胞 (drug-loaded polymeric vesicles) 被成功開發出來。本研究使用 DSA 組成佔高分子總單元數 9 和 17 mol%的含脂質高分子製備載藥液胞，液胞的藥物裝載和釋放與液胞結構對環境pH應答具明顯關聯性。液胞裝載的藥物是帶正電化療藥物doxorubicin (DOX)，利用液胞膜內大量離子化 AAc 單元與 DOX 分子以靜電荷交互作用力彼此吸附，藥物能有效裝載於奈米液胞內 (藥物裝載效率大於85%)。藥物的釋放則是受 AAc/DOX 複合物於不同 pH 環境的穩定度所調控，於 pH 7.4 模擬生理環境中，液胞內大量離子化 AAc 單元能有效與 DOX 分子複合 (complex)，抑制藥物釋放；於pH 5.0弱酸環境中 (如 endosome 或lysosome內)，AAc 與 DOX 間的複合，則會隨著AAc單元質子化而被破壞，藥物會快速從液胞內釋出。由於開發的載藥液胞能藉由液胞外部少量伸展的 DSA 單元，經由 lipid raft-mediated endocytosis 快速進入癌細胞 (HeLa cells)，並於細胞內偏弱酸環境的胞器，如 endosome 或 lysosome 內，迅速釋放藥物引發癌細胞凋亡。
為了提高藥物的傳遞效率及達到即時監控診斷之目的，我們開發出兼具酸鹼調節釋藥 (pH-regulated drug release) 與高度核磁共振影像 (MRI) 對比的裝載 DOX 及 SPIONs 的高分子複合液胞。開發的複合液胞不僅具備奈米尺寸 (約 200 nm)，藉由幾丁聚醣及聚胜肽改質高分子 poly(γ-glutamic acid-co-oxysuccinimidyl glutamate)-g-poly(ethylene glycol)-folate，於高分子液胞表面形成的水膠殼層，液胞的穩定性可被進一步提升，不僅能抵抗大量稀釋及蛋白質的吸附，亦仍保有優異的酸鹼調節藥物釋放特性。由於液胞表面修飾有葉酸，能大幅提高載藥液胞對葉酸受器過度表現的癌細胞 (如 HeLa cells) 的靶向性。另一方面，相較於商品化的核磁共振造影劑 Resovist，本研究開發的裝載 DOX 及 SPIONs 的高分子複合液胞則展現出較佳的核磁共振影像對比性，並且可透過高頻交流磁場的施加，提高載藥液胞的釋藥速率。於動物腫瘤模型的研究則證實，相較於單純使用藥物治療的組別，使用載藥液胞，搭配高頻交流磁場的施予，更能有效地抑制腫瘤生長，說明載藥液胞具備高度開發潛力，可作為新一代多功能化療藥物傳遞系統。
惡性腫瘤深層缺氧區域由於缺乏血管與血液流通，無法有效透過血液傳遞藥物。為突破此困境，具缺氧趨化性的骨髓衍生單核球被用來作為高分子氣/液胞的傳遞媒介，開發出結合活體影像追蹤、癌症治療及超音波控制藥物釋放的多功能藥物傳遞系統。利用含脂質高分子 poly(AAc-co-DSA) 為材料，分別開發出奈米高分子液胞與氣胞，做為化療藥物 DOX 及兼具低沸點及高度超音波對比性之全氟戊烷的搭載平台。載藥液胞能有效抑制藥物於單核球內釋放，但若對裝載高分子氣/液胞的單核球施予聚焦式超音波，氣胞於單核白血球內爆破，產生的剪切力會破壞載藥液胞，促使藥物從單核球釋出，並傳遞至單核球周遭的癌細胞，引發癌細胞凋亡。於本研究中，載藥單核球不僅被證實對動物腫瘤模型具備良好的靶向性與抑制腫瘤生長，甚至能改善經放射治療後的腫瘤復發的情形發生。經腫瘤切片影像證實，載藥單核球不僅能將藥物傳遞至傳統化療藥物或載藥奈米粒子傳遞不彰的固體腫瘤深層缺氧區，甚至是經放射線照射後的腫瘤 (腫瘤內血管密度大幅降低)，亦能有效傳遞藥物並抑制其復發。
In this study, the functionalized micro- and nano-scaled polymer vesicles were successfully developed by the self-assembly of poly(acrylic acid-co-distearin acrylate) (poly(AAc-co-DSA)) with various distearin contents. In order to improve the therapeutic efficacy of cancer treatment, these polymeric vesicles were futher exploited as a vehicle for selective co-delivery of theapeutic and diagnostic agents to the tumor tissues. Notably, through the combination of tumor-homing monocytes with theranostic polymeric assemblies, the effective delivery of therapeutic payloads to tumor hypoxic regions could be realized.
The micro-sized polymer vesicles obtained from self-assembly of poly(AAc-co-DSA) with various distearin contents in aqueous phase show the capability of control over the vesicular-wall permeability to hydrophilic solutes of varying sizes by a simple manipulation of the external pH. The pH sensitivity of the vesicle membranes in size-selective permeability is largely dependent upon the lipid content of copolymer. By the addition of CaCl2 in aqueous vesicle suspensions, the pH-evolved assembly structure and membrane permeability can be immobilized with promoted resistance to further pH alteration, along with an additional counterion screening effect that reduces the pH required for the onset of polar solutes of certain sizes to pass through the membranes. Small angle X-ray scattering measurements of the vesicle structure in the aqueous phase indicate that the pH-regulated permeability to polar solutes is virtually governed by the extent of hydration and swelling of the vesicle membranes.
In addition to the micro-scaled polymeric vesicles, the nano-scaled polymer vesicles were also obtained by self-assembly of poly(AAc-co-DSA) with varying ratios of AAc and DSA units in aqueous solution of pH 5.0 exhibit the pH-regulated drug release behavior. Through the electrostatic interaction with ionized AAc residues, doxorubicin (DOX) molecules can be highly accommodated onto either the inner or outer surfaces of vesicles when the pH is adjusted from 5.0 to 7.4. The extent of DOX encapsulation is dependent largely on the structural transition of vesicles in response to the pH change. While the pH-evolved drug release profile varies to some extent with the distribution of DOX molecules within vesicles, the drug release from vesicles is accelerated significantly via the disruption of the electrostatic interaction of DOX species with ionized AAc moieties at pH 5.0. The DOX-loaded polymer vesicles show promoted cellular uptake and cytotoxicity comparable to free DOX for HeLa cells. This indicates that they are probably taken up by the cells via the lipid raft-mediated endocytosis.
On the other hand, we proposed a novel multifunctional tumor-targeting nanogel-caged polymersome (NCP) carrier system capable of delivering magnetic resonance imaging (MRI) and chemotherapy. Through the self-assembly of poly(AAc-co-DSA) in aqueous suspension of SPIONs and the subsequent DOX encapsulation via electrostatic attraction, the SPION/DOX-loaded polymersomes were attained. Upon sequential deposition of chitosan and poly(gamma-glutamic acid-co-gamma-glutamyl oxysuccinimide)-g-poly(ethylene glycol)-folate (FA) by electrostatic interactions and in situ covalent crosslinking, the NCPs exhibited enhanced excellent anti-dilution, serum proteins-repellent behaviors, pH-tunable drug release and active tumor-targeting ability. The FA-conjugated SPION/DOX-loaded NCPs show superior capability of inhibiting cell proliferation by the combined intracellular pH-triggered rapid drug liberation and magnetic hyperthermia as compared to free DOX species. By virtue of the SPION clustering effect in the inner aqueous chambers of polymersomes, the FA-conjugated SPION/DOX-loaded NCPs have an appreciably higher r2 relaxivity value than Resovist. The high magnetic relaxivity of the tumor-targeting NCPs coupled with enhanced cellular uptake greatly promotes the MRI contrast of target cancer cells. These results demonstrate the great potential of the FA-conjugated SPION/DOX-loaded NCPs as an advanced theranostic nanodevice to improve the anticancer efficacy.
In order to promote the delivery of therapeutic agents toward the hypoxia regions of malignant tumors, an innovative cell-based cancer theranostic system was developed by adopting the tumor-homing monocytes (bone marrow-derived monocytes) as a cellular vehicle for co-delivery of polymer bubbles and DOX-loaded polymeric vesicles. The distearin-conjugated PAAc with the varying DSA contents were used as the major materials to fabricate the nano-scaled polymer vesicles and bubbles. Because of the dense lipid-rich membrane structure, the DOX-encapsulated polymeric vesicles after being internalized by monocytes strongly prevent the drug from leakage, thereby allowing the high activity and viability of monocytes. Moreover, the polymer bubbles within the monocytes still exhibit the long-term profound ultrasound imaging contrast. Interestingly enough, through the focused ultrasound-triggered disruption of polymer bubbles, the generated inertial cavitation most likely impairs the structure of DOX-loaded vesicles, thus facilitating the drug liberation. The in vitro cytotoxicity data further confirm that while being subjected to focused ultrasound treatment and then incubated with TRAMP-C1 cells (murine prostate cancer cells), the payload-containing macrophages displayed a pronounced anticancer efficacy to inhibit cell proliferation. The in vivo antitumor studies also revealed that the tumor growth of tumor-bearing mice with either radiation therapy or not after being intravenously injected with cargo-loaded monocytes and then focused ultrasound treatment was largely inhibited. Besides, the ex vivo histological analyses of tumor sections further proved the successful transport of DOX by monocytes to the tumor hypoxic regions far away from the blood vessels compared to that by polymeric vesicles alone. This work demonstrates that monocyte-based functionalized nanoparticles delivery system in combination with ultrasound-activated drug release open a new opportunity for cancer therapy of tumor hypoxic areas.